Within the field of biological diagnostics, more research and analytical procedures require the investigation of nucleic acids from biological specimens, such as whole blood, serum, semen, biological tissues and other human or animal sources. For diagnostic applications in particular, a targeted nucleic acid sequence may be only a small portion of the DNA or RNA being considered, so that detection of its presence may be very difficult. The well known DNA probe technology is steadily being improved to address this problem, providing more sensitive probes.
However, a significant development in providing highly detectable quantities of nucleic acids is described in U.S. Pat. No. 4,683,195 (issued July 28, 1987 to Mullis et al) and U.S. Pat. No. 4,683,202 (issued July 28, 1987 to Mullis). These references describe the amplification of a targeted nucleic acid sequence using DNA primers and agents for adding bases to DNA strands complementary to the targeted sequence. After a multiplicity of cycles of priming, extending DNA strands and denaturation, the targeted nucleic acid can be more readily detected using labeled primers, probes or other means.
Primer extension is accomplished in the presence of suitable amounts of deoxyribonucleoside triphosphates and an agent for inducing the extension (also called a polymerization agent). Generally, such agent is a DNA polymerase such as an E. coli DNA polymerase I, Klenow polymerase, T4 DNA polymerase and others which will facilitate combination of the deoxyribonucleoside triphosphates in the proper manner to form the primer extension products. Particularly useful enzymes are thermally stable enzymes, cloned or naturally occurring, such as those obtained from various bacterial species, such as the Thermus species. Some useful polymerases are commercially available. Generally, the synthesis of extension products will be initiated at the 3' end of each primer and proceed in the 5' to 3' direction along the template until synthesis is terminated.
Preferred thermally stable enzymes are DNA polymerases from Thermus aquaticus, such as described in EP-A-0 258 017 (published Mar. 2, 1988).
In various instances, it is important to know how much DNA polymerase is present in a medium. It is quite costly to isolate or clone a polymerase, and its efficient use is highly desirable. The amount of polymerase present in a sequencing process should be determinable. In the isolation or cloning of polymerases, it would be desirable to know when the desired concentration of enzyme has been obtained. In addition, it may be necessary to understand the enzymatic capabilities of a given polymerase in a given amplification medium.
Various isolation and purification methods for DNA polymerases are known in the art (including those references noted above). EP-A-0 258 017 (noted above) mentions a number of them. The thermostable enzymes described in that reference are assayed using a tedious six-step procedure described by Kaledin et al, Biokhymiya, 45, pp. 644-651 (1980) involving chromatography, fractionations and other time-consuming techniques.
Another procedure for measuring the amount of a DNA polymerase involves measuring the rate of incorporation of radioactively labeled deoxyribonucleoside triphosphates into a primer extension product (see generally Kornberg, DNA Replication, W. H. Freeman & Co., San Francisco, 1980, Chapters 4-6). However, this procedure likewise has a number of disadvantages, including imprecision and awkwardness. Moreover, the use of radioactive labels is not desired due to the handling and safety hazards involved.
It has been known for some time that certain fluorescent compounds (such as one known in the art as Hoechst 33258 Dye) bind preferentially to double-stranded DNA with a shift in signal as opposed to binding with the single-stranded form. See, for example, Labarca et al, Anal. Biochem., 102, pp. 344-352 (1980), Downs et al, Anal. Biochem., 131, pp. 538-547 (1983), Sterzel et al, Anal. Biochem., 147, pp. 462-467 (1985), Perkin Elmer Technical Bulletin L-913A, Sept., 1986 and Perkin Elmer Cetus bulletin "Amplifications", pp. 8-10, February, 1989. Moreover, some researchers have used the Hoechst 33259 Dye in temperature optimization studies involving polymerase chain reaction. Such studies involved measuring the fluorescent signal obtained at certain time intervals for several polymerization temperatures. However, such studies were not used for, and indeed were incapable of, quantitatively determining the amount of polymerase in a specimen. In such instances, the rate of polymerase reaction was not measurable with accuracy, nor was a correlation established between the measured rate and polymerase activity.
Thus, while many researchers have considered the use of various dyes for detection of DNA, none of them suggests how they can be used to quantitatively determine the amount of a DNA polymerase in a solution. Yet, there is a need in the art for a quantitative, safe and convenient method for assaying for DNA polymerase in an aqueous medium.